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Expression of high-affinity human antibody fragments in bacteria


Here we describe protocols for the expression of human antibody fragments in Escherichia coli. Antigen-specific clones are identified by soluble fragment ELISA and concentrated by periplasmic preparation. They are then further purified by affinity chromatography. This article provides an overview of expression and purification strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.

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Figure 1: Analysis of sample purity by SDS-PAGE.
Figure 2: Analysis of sample purity by size-exclusion chromatography.


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Protocols are based on methods originally developed by D. Christ and others in G. Winter's group at the UK Medical Resarch Council (MRC) Laboratory of Molecular Biology. These were further modified at the Garvan Institute of Medical Research and at MedImmune. This work was funded by the Garvan Institute of Medical Research; MedImmune; the Australian National Health and Medical Council; the Australian Research Council; the Cancer Institute, NSW; and the UK MRC.

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D. Lowe, K.D., B.R., P.S., D. Langley, J.A. and P.W. wrote the paper. R.R. wrote the paper and generated figures. L.J. and D.C. wrote the paper and supervised research.

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Correspondence to Daniel Christ.

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The authors declare no competing financial interests.

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Rouet, R., Lowe, D., Dudgeon, K. et al. Expression of high-affinity human antibody fragments in bacteria. Nat Protoc 7, 364–373 (2012).

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